We are studying molecular mechanisms underlying neuronal targeting using the leech embryo as a model system. Using monoclonal antibodies generated against leech surface antigens, we previously determined that all sensory neurons express a unique mannose-containing epitope. In contrast, different subsets of these same sensory neurons can be differentiated by their complex carbohydrate epitopes containing alpha and beta galactose. Using different experimental manipulations with FAB fragments, enzymes and neoglycoproteins, we demonstrated the involvement of these different carbohydrate epitopes (or markers) in two sequential targeting steps during the development of these sensory neurons. To structurally analyze the mannose and galactose-containing carbohydrate markers, we immunopurified glycoproteins using our monoclonal antibodies. We found that lipids coprecipitate together with these glycoproteins. Therefore, this past year we analyzed the lipid content of the leech, and we also analyzed the lipid environment of the sensory neurons glycoproteins using GC-MS. We found that 5% of the leech lipids consist of cholesterol as well as an unusual steroid. GC-MS data showed that this novel steroid is more highly hydroxylated than cholesterol, a finding that was supported by DQF-COSY. GC-MS data also showed that leech glycolipids may be unusual in that they are rich in xylose and dideoxyhexoses. Having completed the GC-MS analysis of leech lipids, we are now planning to pursue our original goal, namely the GC-MS analysis of the carbohydrate markers that mediate the two targeting steps of sensory neurons.